CN101213293A - Microorganism of enterobacteriacae genus haboring genes associated with L-carnitine biosynthesis and method of producing L-carnitine using the microorganism - Google Patents
Microorganism of enterobacteriacae genus haboring genes associated with L-carnitine biosynthesis and method of producing L-carnitine using the microorganism Download PDFInfo
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Abstract
Provided is a microorganism that belongs to Enterobacteriacae and a method of producing L-carnitine using the same. The microorganism includes polynucleotide encoding activity of S-adenosylmethionine-6-N-iysine metyltransferase from Neurospora crassa, polynucleotide encoding activity of 6-N-trimethyllysine hydroxylase, polynucleotide encoding activity of 3-hydroxy-6-N-trimethyllysine aldolase, and polynucleotide encoding activity of Gamma-trimethylaminoaldehyde dehydrogenase and Gamma-butyrobetaine hydroxylase.
Description
Technical field
The present invention relates to comprise the L-carnitine biosynthesis related genes that derives from Neuraspora crassa (Neurospora crassa) enterobacteriaceae (Enterobacteriacae) microorganism and use the method for described microorganisms producing L-carnitine.
Background technology
L-carnitine (3-hydroxyl-4-trimethylammonium aminobutyric acid) ubiquity in organism, and be in plastosome, the activatory longer chain fatty acid to be carried to zwitterionic compound in the mitochondrial matrix via mitochondrial inner membrane.It is known that the L-carnitine can be synthetic from Methionin or protein Methionin in human body.In Mammals, protein Methionin is used as the biosynthetic precursor of L-carnitine usually, yet, in Neuraspora crassa, use free Methionin.In the biosynthesizing of L-carnitine, form ε-N, N, N-trimethyl lysine, ε-N, N, N-trimethylammonium-beta-hydroxy Methionin, N, N, N-trimethylammonium amino butyraldehyde intermediate, and gamma-butyrobetaine, and gamma-butyrobetaine becomes the L-carnitine by the gamma-butyrobetaine hydroxylase hydroxylation.Fig. 1 is the schema of the L-carnitine biosynthetic pathway supposed in the diagram Neuraspora crassa.
The L-carnitine can pass through chemical synthesis, the semi-synthesis method of using enzyme reaction and the method production of using microbe.Yet when the applied chemistry synthesis method, existing problems promptly obtain DL-carnitine racemic mixture, must separate the DL-racemic mixture thus.As an example of the semi-synthesis method of using enzyme reaction, U.S. Patent number 4,221,869 disclose the carnitin dehydrogenases (EC 1.1.1.108) of NAD as coenzyme, the method for being produced the L-carnitine by the dehydrogenation carnitine used of using.Yet, extremely unstable and spontaneous acetonyl TMA (TriMethylAmine) and the carbonic acid gas of being decomposed into of dehydrogenation carnitine.In addition, German patent DE-OS-3123975 discloses the method for being produced the L-carnitine by gamma-butyrobetaine, and this method is used from the isolating gamma-butyrobetaine hydroxylase of Neuraspora crassa (EC 1.14.11.1).Yet, there is a shortcoming, promptly during hydroxylation, α-Tong Wuersuan and reduzate (that is xitix) must be added in the reactant.
Produce the method for L-carnitine about using microbe, for example, U.S. Patent number 5,028,538 disclose and are a kind ofly containing Ba Dousuan trimethyl-glycine (4-N from intestinal bacteria (E.coli) 044 K 74 is cultivated, N, the amino Ba Dousuan of N-triethyl) culture that obtains after the substratum and the method for collecting the L-carnitine.In addition, U.S. Patent number 4,708,936 disclose by Alcaligenes xylosoxidans (Achromobacter xylosoxydans) DSM 3225 (HK 1331b) being cultivated the method for producing the L-carnitine in the substratum that contains Ba Dousuan trimethyl-glycine and/or gamma-butyrobetaine.Yet, there is shortcoming, that is, should use the biosynthetic precursor of L-carnitine, such as the Ba Dousuan trimethyl-glycine, or be not the compound of intermediate, and the productive rate of L-carnitine is not high.Therefore, in the method for using microbe production L-carnitine, still there are the needs that improve productive rate.
The present inventor has attempted the production application inexpensive precursors and has had the microorganism of the L-carnitine of high yield, and find that deriving from the Neuraspora crassa gene relevant with the biosynthesizing of L-carnitine gives full expression to, and finishes the present invention thus in the microorganism of enterobacteriaceae.
Accompanying drawing is described
Fig. 1 is the schema of the L-carnitine biosynthetic pathway supposed in the diagram Neuraspora crassa.
Fig. 2 is the result's that shows that the natural or SDS-PAGE of elute soln analyzes figure, and described elute soln is by cracking Neuraspora crassa culture and the cracked material is carried out the DEAE column chromatography obtain.
Fig. 3 is protein band a, b and c and Methionin and the back chart that detects the result of trimethyl lysine by HPLC of S-adenosylmethionine reaction that is presented at Fig. 2.
Fig. 4 shows by HPLC to analyze chart by the result of sample that band a albumen test is obtained and trimethyl lysine standard substance.
Fig. 5 is the figure that shows by the electrophoresis result of pcr amplification LMT gene.
Fig. 6 illustrates the production process of pT7-7 LMT.
Fig. 7 is the figure that shows the supernatant liquor SDS-PAGE analytical results of cracked bacterium, and described cracked bacterium contains the intestinal bacteria of the S-adenosylmethionine-6-N-Methionin-methyltransgerase that derives from Neuraspora crassa by cultivation in the presence of IPTG and the bacterium cracking that will therefrom obtain obtains.
Fig. 8 illustrates the production process of pT7-7 TMLH.
Fig. 9 illustrates the production process of pT7-7 TMLA.
Figure 10 illustrates the production process of pT7-7TMABADH.
Figure 11 illustrates the production process of pT7-7 BBH.
Figure 12 is the photo that shows the electrophoresis result that is inserted into each gene among pT7-7 TMLH, pT7-7 TMLA, pT7-7TMABADH and the pT7-7 BBH respectively.In Figure 12, swimming lane 1 is represented mark, and swimming lane 2 is represented pT7-7 TMLH, and swimming lane 3 is represented pT7-7 TMLA, and swimming lane 4 is represented pT7-7TMABADH, and swimming lane 5 is represented pT7-7 BBH.
Figure 13 is the photo that shows the SDS-PAGE result of crude extract, and described crude extract is respectively available from the culture of the e. coli bl21 (DE3) that transforms with pT7-7 TMLH, pT7-7 TMLA, pT7-7TMABADH and pT7-7 BBH.In Figure 13, swimming lane 1 is represented mark, and swimming lane 2 is represented negative control group, and swimming lane 3 is represented pT7-7TMLH (52kDa), and swimming lane 4 is represented pT7-7TMLA (53kDa), and on behalf of pT7-7TMABADH (55kDa) and swimming lane 6, swimming lane 5 represent pT7-7BBH (49kDa).
Figure 14 illustrates the production process of pT7-7CarABE.
Figure 15 illustrates the production process of pACYC184CarCD.
Detailed Description Of The Invention
Technical problem
The invention provides can high efficiency production L-carnitine microorganism.
The present invention also provides the method for using described microorganisms producing L-carnitine.
Technical scheme
According to one aspect of the present invention, a kind of microorganism that belongs to enterobacteriaceae is provided, described microorganism comprises: the S-adenosylmethionine-active polynucleotide of 6-N-Methionin-methyltransgerase (LMT) of coding source Neuraspora crassa; The coding 3-hydroxyl-active polynucleotide of 6-N-trimethyl lysine zymohexase (TMLA); The active polynucleotide of coding N-trimethyl lysine hydroxylase (TMLH); Coding γ-active polynucleotide of trimethylammonium amino-aldehyde desaturase (TMABADH); With the active polynucleotide of coding gamma-butyrobetaine hydroxylase (BBH).
According to microorganism of the present invention can be contain the coding described 5 kinds of proteic polynucleotide any.Preferably, described microorganism is intestinal bacteria (Escherichia coli), and more preferably intestinal bacteria (preserving number: KCCM-10638).
According to 5 kinds of albumen of absolute coding of the present invention, that is, the polynucleotide of LMT, TMLH, TMLA, TMABADH and BBH can be used for microorganism by carrier or itself.When the described 5 kinds of proteic polynucleotide of absolute coding are used for microorganism by carrier, the described 5 kinds of proteic polynucleotide of coding can be inserted in the single carrier and use thereafter, perhaps can be inserted at least a carrier and use thereafter.In the present invention, term " carrier " is that those skilled in the art is known.Carrier typically refers to the nucleic acid construct that is used for nucleic acid is introduced cell.This nucleic acid construct can be to be derived from plasmid or virus genomic nucleic acid construct.
The polynucleotide encoding that derives from the S-adenosylmethionine-6-N-Methionin-methyltransgerase (LMT) of Neuraspora crassa according to one embodiment of the invention coding derives from the S-adenosylmethionine lysine methyltransferase of Neuraspora crassa.Think that the catalysis in the Neuraspora crassa cell of S-adenosylmethionine lysine methyltransferase transforms into the reaction of 6-N-trimethyl lysine by methyl group being attached on the Methionin with Methionin, but scope of the present invention is not limited to this concrete mechanism of action.The polynucleotide of coding S-adenosylmethionine lysine methyltransferase are the polynucleotide of encoding amino acid sequence SEQ ID NO:11 preferably, and more preferably are the polynucleotide with nucleotide sequence SEQ ID NO:16.
The polynucleotide encoding that derives from the N-trimethyl lysine hydroxylase (TMLH) of Neuraspora crassa according to one embodiment of the invention codings derives from the N-trimethyl lysine hydroxylase (TMLH) of Neuraspora crassa.Think that N-trimethyl lysine hydroxylase (TMLH) catalysis in the Neuraspora crassa cell changes into the reaction of beta-hydroxy-ε-N-trimethyl lysine with the N-trimethyl lysine, but scope of the present invention is not limited to this concrete mechanism of action.The polynucleotide of coding N-trimethyl lysine hydroxylase (TMLH) are the polynucleotide of encoding amino acid sequence SEQ ID NO:12 preferably, and more preferably have the polynucleotide of nucleotide sequence SEQ ID NO:17.
The polynucleotide encoding that derives from the 3-hydroxyl-6-N-trimethyl lysine zymohexase (TMLA) of Neuraspora crassa according to one embodiment of the invention codings derives from the 3-hydroxyl-6-N-trimethyl lysine zymohexase (TMLA) of Neuraspora crassa.Think that the catalysis in the Neuraspora crassa cell of 3-hydroxyl-6-N-trimethyl lysine zymohexase (TMLA) changes into the reaction of γ-N-trimethylammonium amino butyraldehyde with beta-hydroxy-ε-N-trimethyl lysine, but scope of the present invention is not limited to this concrete mechanism of action.The polynucleotide of coding 3-hydroxyl-6-N-trimethyl lysine zymohexase (TMLA) are the polynucleotide of encoding amino acid sequence SEQ ID NO:13 preferably, and more preferably have the polynucleotide of nucleotide sequence SEQ ID NO:18.
γ-active the polynucleotide encoding of trimethylammonium amino-aldehyde desaturase (TMABADH) that derives from Neuraspora crassa according to one embodiment of the invention codings derives from γ-trimethylammonium amino-aldehyde desaturase (TMABADH) activity of Neuraspora crassa.Think that the catalysis in the Neuraspora crassa cell of γ-trimethylammonium amino-aldehyde desaturase (TMABADH) changes into the reaction of gamma-butyrobetaine with γ-N-trimethylammonium amino butyraldehyde, but scope of the present invention is not limited to this concrete mechanism of action.The polynucleotide of coding γ-trimethylammonium amino-aldehyde desaturase (TMABADH) are the polynucleotide of encoding amino acid sequence SEQ ID NO:14 preferably, and more preferably have the polynucleotide of nucleotide sequence SEQ ID NO:19.
The active polynucleotide encoding of gamma-butyrobetaine hydroxylase (BBH) that derives from Neuraspora crassa according to one embodiment of the invention codings derives from the gamma-butyrobetaine hydroxylase (BBH) of Neuraspora crassa.Think that gamma-butyrobetaine hydroxylase (BBH) catalysis in the Neuraspora crassa cell changes into the reaction of L-carnitine with gamma-butyrobetaine, but scope of the present invention is not limited to this concrete mechanism of action.The polynucleotide of coding gamma-butyrobetaine hydroxylase (BBH) are the polynucleotide of encoding amino acid sequence SEQ ID NO:15 preferably, and more preferably are the polynucleotide with nucleotide sequence SEQID NO:20.
According to another aspect of the present invention, the method of producing the L-carnitine is provided, described method comprises: in the presence of substrate, cultivate according to microorganism of the present invention, to produce the L-carnitine in culture, described substrate is selected from the group of being made up of following: L-Methionin, the N-trimethyl lysine, beta-hydroxy-N-trimethyl lysine, γ-N-trimethylammonium amino butyraldehyde, gamma-butyrobetaine, and composition thereof.
Producing according to the present invention in the method for L-carnitine, the concentration that is selected from the substrate of the group of being made up of L-Methionin, N-trimethyl lysine, beta-hydroxy-N-trimethyl lysine, γ-N-trimethylammonium amino butyraldehyde, gamma-butyrobetaine and composition thereof is preferably the 0.1-10 weight % based on substratum weight, but the present invention is not particularly limited to this scope.
In according to method of the present invention, be that those skilled in the art is known from the method for culture collection L-carnitine.The example of such method comprises, but be not limited to, ultrafiltration, centrifugation with at method such as the decantation of cell being collected the L-carnitine from the product recrystallization that obtains after culture separates, and carry out cation-exchange chromatography or electrodialysis for the supernatant liquor that therefrom obtains.
Advantageous effects
Have the good capacity of producing the L-carnitine according to microorganism of the present invention, so that it can be effective to the method by fermentative production L-carnitine.
Producing according to the present invention in the method for L-carnitine, using the microorganism that belongs to enterobacteriaceae can produce the L-carnitine efficiently.
Best mode
Below, will the present invention more specifically be described with reference to the following example.These embodiment only are that illustration purpose is not that intention limits the scope of the invention for example.
Embodiment
Generation be coded in the Neuraspora crassa with by 5 kinds of relevant proteic polynucleotide of L-Methionin biosynthesizing L-carnitine, and the nucleic acid construct that comprises it.Then, with described nucleic acid construct transformed into escherichia coli, and the intestinal bacteria that transform are cultivated in the substratum that contains the intermediate product that obtains by L-carnitine production approach, to produce the L-carnitine and to collect the L-carnitine.
Embodiment 1: LMT, the TMLH that separating encodes derives from Neuraspora crassa, TMLA,
The polynucleotide of TMABADH and BBH
Separate and clone the polynucleotide of coding LMT, the TMLH, TMLA, TMABADH and the BBH that derive from Neuraspora crassa, and analyze its base sequence.
(1) the cDNA library of preparation Neuraspora crassa
Separate total mRNA from the culture that comprises Neuraspora crassa fungus body (comprising sporophyte), and use poly T to carry out reverse transcription, carry out pcr amplification cDNA then as primer.The cDNA of amplification is digested with EcoR I and Xho I, then the cDNA of digestion is inserted into the EcoR I and the Xho I site of λ AD5 cloning vector, derive from the cDNA library of Neuraspora crassa with preparation.
Then, with described cDNA library ehec infection BNN322, cultivate then and intestinal bacteria BNN322 that amplification is infected.At first, with intestinal bacteria BNN322 overnight incubation in the LB substratum that contains 50 μ g/ml kantlex and 0.2% maltose.Then, carry out centrifugation, remove the supernatant liquor of the product that obtains then, and afterwards cell precipitation is resuspended in 1ml 10mM MgSO for culture from its acquisition
4In the solution.The suspension and 5 * 10 that will obtain from the product that obtains
7The λ cDNA library of PFU adds 2ml LB substratum in addition 30 ℃ of nonoscillatory ground incubations 30 minutes in culture, and the culture that then will obtain is 30 ℃ of vibrations 1 hour in the shaking culture case.Cultured cells is scoring on the LB culture plate that contains penbritin (75 μ g/ml), and cultivated 8 hours at 37 ℃.Use the Wizard test kit to separate cDNA library set (cDNA library pool) from the bacterium colony of flat board.The λ that will comprise the set of isolating cDNA library is as template, with the polynucleotide of amplification coding LMT, TMLH, TMLA, TMABADH and BBH.
(2) polynucleotide (LMT gene) of amplification and clones coding LMT and confirmation LMT
Produce
(a) separate the LMT gene and confirm the functional expression of described gene from Neuraspora crassa
Cultivate Neuraspora crassa and collecting cell.Then, the potassium phosphate buffer that uses the 1M pH 7.4 that contains 2mM DTT and 0.2mMEDTA extracts albumen then with lysis.Ammonium sulfate is slowly joined in the supernatant liquor that is obtained, reach 50% whole saturation concentration and albumen precipitation is got off, add the potassium phosphate buffer of a small amount of 0.1M pH 7.4 then to albumen by centrifugation.Use the T1 dialysis membrane to carry out desalination the solution that obtains, and the sample of desalination is used the DEAE column purification.At this moment, use the potassium phosphate buffer of 0.1M pH 7.4 to merge (pooling) as elution buffer as lavation buffer solution and the potassium phosphate buffer that comprises the 0.1M pH 7.4 of 0.3M NaCl.After this, the sample that merges is used the desalination of T1 dialysis membrane.The desalination sample uses the CM column purification.With the potassium phosphate buffer of 0.1M pH 7.4 lavation buffer solution as described post, and will not be adsorbed onto on the post and from the post effusive sample all merge.
Protein sample is loaded on the DEAE post once more, uses the potassium phosphate buffer of 0.1M pH 7.4 then, carry out the density gradient wash-out, reach the NACl concentration of 0-0.3M.Use natural-PAGE and SDS-PAGE that the sample of purifying is carried out protein analysis.
Fig. 2 shows the natural-PAGE of elute soln or the result's that SDS-PAGE analyzes figure, and the solution of described wash-out obtains after carrying out the DEAE column chromatography with the cracking of Neuraspora crassa culture and to institute's cracked material.In Fig. 2, swimming lane 1 is represented mark, the result that on behalf of natural-PAGE of DEAE elution peak 2, swimming lane 2 and 3 analyze, and swimming lane 4 and 5 is represented the result of natural-PAGE analysis of DEAE elution peak 3.In Fig. 2 B, swimming lane 1 is represented mark, the result that on behalf of the natural-PAGE of DEAE elution peak 2, swimming lane 2 analyze, the result that on behalf of the natural-PAGE of DEAE elution peak 3, swimming lane 3 analyze, the result that on behalf of the SDS-PAGE of DEAE elution peak 2, swimming lane 4 and 5 analyze, and swimming lane 6 and 7 is represented the result of the SDS-PAGE analysis of DEAE elution peak 3.
From the result of Fig. 2, band a, b and c are elected to be the LMT candidate albumen, and measure each proteic activity.At first, gel that will be corresponding with each band downcuts, and with homogenizer gel is crushed then.Then, to wherein adding 5ml 1g/L Methionin (final concentration 500mg/L) and 2ml 1g/L methyl donor, S-adenosylmethionine (final concentration 200mg/L), and the product that obtains slowly stirred at 28 ℃ reacted in 24 hours, use HPLC to analyze the trimethyl lysine peak then.
Fig. 3 is presented at protein band a, b and c and Methionin and the back chart of measuring the result of trimethyl lysine by HPLC of S-adenosylmethionine reaction.As shown in FIG. 3, with the sample of band a reaction in, turn out to be the peak that is regarded as trimethyl lysine in about 15 minutes residence time.In Fig. 3,1,2 and 3 representatives and each band a, b and the corresponding result of c.In order to confirm described band exactly, the sample and the trimethyl lysine standard substance that will obtain by the reaction with band a compare.
Fig. 4 shows by HPLC to analyze chart by the result of the sample that obtains with protein band a reaction and trimethyl lysine standard substance.As shown in FIG. 4, during the peak of band a, that is, voltage has the time of maximum, and is in full accord with standard trimethyl lysine sample.Therefore, confirm that band a comprises S-adenosylmethionine-6-N-Methionin-methyltransgerase, LMT.In Fig. 4,1 and 2 are meant respectively and standard substance and the corresponding result of band a.Fig. 2 and each chart of 3 are that independently the HPLC chart is incorporated into wherein chart.
Next, analyze the N-terminal sequence, to obtain the proteic aminoacid sequence of LMT.At first, will transfer on the pvdf membrane, then protein band be downcut, to pass through Edman methods analyst N-terminal sequence at the albumen on the SDS-PAGE gel.Especially, thiocarbanil (PTC) and peptide are at pH value 8-9 and room temperature reaction, and therefore obtaining wherein, N-holds by the PTC-peptide of thiocarbamoylization.Then, the PTC-peptide is reacted under acidic conditions, with only isolating n terminal amino acid therefrom.Isolating amino acid ethyl acetate extraction is identified with HPLC, and analyzes.As a result, confirm that the N-terminal sequence is AFGKL (SEQ ID NO:21).Similarly, carry out seeking based on the n terminal amino acid sequence that is confirmed about the complete genome sequence of known Neuraspora crassa.As a result, confirm to have albumen and the gene and the nucleotide sequence of the aminoacid sequence consistent with the N-terminal sequence of LMT.
(b) carry LMT expression carrier and microorganisms producing
Collect the Neuraspora crassa of cultivating, and use the liquid nitrogen cracking, use RNA purification kit purifying RNA then.Application is produced the primer of SEQ ID NOS:1 and 2 about the information of the amino acid of the LMT that confirms and base sequence in (a), then, the cDNA library that use produces in (1) is by using described primer to the S-adenosylmethionine-6-N-Methionin-methyl transferase gene (Fig. 5) that increases of the PCR as primer.Fig. 5 is the figure that shows the electrophoresis result of the LMT gene that passes through pcr amplification.
The PCR product and the pT7-7 carrier that obtain are digested with Nde I and BamH I respectively, and use the T4 dna ligase to be connected to each other, to produce pT7-7 LMT carrier (Fig. 6).Fig. 6 illustrates the production process of pT7-7 LMT.Use electroporation pT7-7 LMT carrier transformed into escherichia coli BL21 DE3.The e. coli bl21 DE3 of 40 μ l and the pT7-7 LMT carrier of 1 μ l are mixed, place ice-cold cuvette, and, transform by electroporation under the condition of 200 Ω and 25 μ F at 2.5kV with 2mm slit.The transformant that obtains is scoring on the solid plate substratum that contains penbritin, then with plasmid from selected transformant purifying therein, and digest with NdeI and BamH I.As a result, by the gene of confirmation insertion and the size of plasmid, and confirm that pT7-7LMT is incorporated in the plasmid; This is called BL21 (DE3)/pT7-7LMT.
(c) expression in escherichia coli S-adenosylmethionine-6-N-Methionin-methyltransgerase and
Produce trimethyl lysine from Methionin
BL21 (DE3)/pT7-7LMT is cultured to OD in the LB substratum
6000.5, after wherein adding 1mM IPTG, cultivating again 4 hours then.Culture is carried out centrifugal, and collecting cell and use ultrasonic treatment.Carry out SDS-PAGE by the pair cell lysate, confirm S-adenosylmethionine-6-N-Methionin-methyltransgerase (Fig. 7) of about 25kD.Fig. 7 is the figure that shows the SDS-PAGE analytical results of supernatant liquor, and wherein said supernatant liquor contains the intestinal bacteria of the S-adenosylmethionine-6-N-Methionin-methyltransgerase that derives from Neuraspora crassa by cultivation in the presence of IPTG and the microbial lytic that will therefrom obtain obtains.In Fig. 7, swimming lane M is a digit synbol, and swimming lane 1 is meant negative control group, and swimming lane 2 and 3 is meant cell lysate, and the annular section in swimming lane 2 and 3 is meant at 25 kD positions and the corresponding band of LMT.
E. coli bl21 (DE3)/pT7-7LMT is equipped with therein in the 250ml flask that baffle plate is housed of the LB substratum that contains the 50ml penbritin and is cultured to OD
6000.6, then after wherein adding 1mM IPTG, cultivate to surpass 8 hours again at 28 ℃, forming the correct tertiary structure of enzyme, and prevent the formation of inclusion body.In culturing process, the L-Methionin of adding 500mg/L and the S-adenosylmethionine of 200mg/L are as reaction soln, and the trimethyl lysine content in the measurement culture solution.The result is displayed in Table 1.
Measure trimethyl lysine by HPLC under the following conditions.The SUPELCOSIL LC-DABS that derives from Supelco is as post.The A damping fluid prepares like this, so that 0.1% trifluoroacetic acid (TFA) is joined wherein distilled water and acetonitrile with in the mixed damping fluid of 2: 8 ratios, and the B damping fluid prepares like this, so that 0.1% TFA is joined wherein distilled water and acetonitrile with in the mixed damping fluid of 2: 8 ratio.Use the linear concentration gradient method, keep the flow velocity of 0.8ml/min, analyze trimethyl lysine.
Table 1.
| The material of measuring | Trimethyl lysine (μ g/ml) |
| E. coli bl21 (DE3)/pT7-7 (IPTG induces)+500mg/L Methionin+200mg/L Ado-Met | 0.0 |
| E. coli bl21 (DE3)/pT7-7LMT (IPTG induces)+500mg/L Methionin+200mg/LAdo-Met | 20.0 |
As shown in the table 1, the gene of S-adenosylmethionine-6-N-Methionin-methyltransgerase that confirms to derive from Neuraspora crassa is at expression in escherichia coli, and therefrom L-Methionin changed into trimethyl lysine.
(3) polynucleotide (TMLH gene) of amplification and clones coding TMLH, and confirm
The generation of TMLH
(a) polynucleotide (TMLH gene) of amplification and clones coding TMLH
Use comprises the λ of cDNA library set of (1) as template, and uses SEQ ID NOS:3 and 4 to carry out PCR as primer.Then, carry out agarose gel electrophoresis for the PCR product that obtains.As a result, confirm the product that needs of about 1.4kb.SEQ ID NOS:3 and 4 primer comprise that the supposition coding derives from the initiator codon of TMLH of Neuraspora crassa and the sequence of terminator codon.By from the aminoacid sequence of the whole protein of Neuraspora crassa genomic expression and derive from the people and the aminoacid sequence of the known TMLH of rat between carry out the potential TMLH that homology search derives from Neuraspora crassa, from the aminoacid sequence design SEQ ID NOS:3 of potential TMLH and 4 primer.
With the PCR product with EcoRI and SalI digestion, and with pBS KS with identical enzymic digestion
+(Stratagene Inc.) connects, then with the bacillus coli DH 5 alpha pBS KS that wherein inserts the PCR product that is obtained
+(TMLH) transform.The bacillus coli DH 5 alpha that transforms was cultivated 8 hours at 37 ℃, separated pBS KS then
+And, whether correctly insert (TMLH), to determine the PCR product with EcoRI and SalI digestion.Then, with isolating pBS KS
+(TMLH) with NdeI and SalI digestion, after agarose gel electrophoresis, NdeI is separated with the SalI fragment then.With described fragment with connect with the expression vector pT7-7 of identical enzymic digestion, to obtain pT7-7 TMLH (referring to Fig. 8).PT7-7 (TMLH) is transformed in the e. coli bl21 (DE3).
(b) confirm that TMLH produces
The e. coli bl21 (DE3) that will transform with the pT7-7 (TMLH) that obtains is cultured to OD in 37 ℃ of 250ml flasks that baffle plate is housed that are equipped with the 50ml LB substratum that contains 100 μ g/ml penbritins therein
6000.6, and after wherein adding 1mM IPTG, continuing cultivation 4 hours.PT7-7 (TMLH) is separated from culture, and, carry out agarose gel electrophoresis then with NdeI and SalI digestion.The result shows in Figure 12.As shown in Figure 12, confirmation and NdeI and the corresponding band of SalI fragment (swimming lane 2).Then, separate pT7-7 (TMLH), and analyze the nucleotide sequence of TMLH.As a result, the nucleotide sequence of TMLH confirms it is the sequence (SEQ ID NO:17) identical with the sequence of preserving in the Neuraspora crassa genome database of NCBI.
In addition, in the culture of the e. coli bl21 (DE3) that transforms with pT7-7 (TMLH), confirm the TMLH albumen of expression.At first, 4, centrifugal 15 minutes of 000xg carries out centrifugation to culture, and the collecting cell precipitation.The lysis buffer (140mM NaCl, 200g/l glycerine and 1mM DTT are in the buffer solution of sodium phosphate of 10mM pH 7.4) of adding 1ml and resuspended in the cell precipitation that obtains.Cell suspension is placed ice bath, and cell is used the sonicator cracking by transmitting ultrasonic wave 5 times each 10 seconds.The pair cell lysate carries out centrifugation, at 4 ℃, and 10, the centrifugal 20-30 of 000g minute, remove cell debris then, and collect supernatant liquor to obtain cell crude extract.The sample that comes from the cell crude extract of acquisition by collecting carries out 8%SDS-PAGE (referring to Figure 13, swimming lane 2).As the result who carries out SDS-PAGE, confirm band with the corresponding about 52kDa of TMLH.
(3) amplification and clones coding 3-hydroxyl-6-N-trimethyl lysine zymohexase (TMLA) is many
The generation of Nucleotide and confirmation TMLA
(a) amplification and clones coding 3-hydroxyl-6-N-trimethyl lysine zymohexase (TMLA) is many
Nucleotide
Use comprises the λ of cDNA library set of (1) as template, and uses SEQ ID NOS:5 and 6 to carry out PCR as primer.Then, carry out agarose gel electrophoresis for the PCR product that obtains.As a result, confirm the product that needs of about 1.4kb.SEQ ID NOS:5 and 6 primer comprise that coding derives from the initiator codon of TMLA of Neuraspora crassa and the sequence of terminator codon.By from the aminoacid sequence of the whole protein of Neuraspora crassa genomic expression and derive from the people and the aminoacid sequence of the known TMLA of rat between carry out the potential TMLA that homology search derives from Neuraspora crassa, from the aminoacid sequence design primer SEQ IDNOS:5 and 6 of potential TMLA.
With the PCR product with EcoRI and SalI digestion, and with pBS KS with identical enzymic digestion
+(Stratagene Inc.) connects, then with the bacillus coli DH 5 alpha pBS KS that wherein inserts the PCR product that is obtained
+(TMLA) transform.The bacillus coli DH 5 alpha that transforms was cultivated 8 hours at 37 ℃, therefrom separated pBS KS then
+And, whether correctly insert (TMLA), to determine the PCR product with EcoRI and SalI digestion.Then, with isolating pBS KS
+(TMLA) with NdeI and SalI digestion, after agarose gel electrophoresis, NdeI is separated with the SalI fragment then.With described fragment with connect with the expression vector pT7-7 of identical enzymic digestion, to obtain pT7-7 (TMLA) (referring to Fig. 9).E. coli bl21 (DE3) is transformed with pT7-7 (TMLA).
(b) confirm that TMLA produces
The e. coli bl21 (DE3) that will transform with the pT7-7 (TMLA) that obtains is cultured to OD in 37 ℃ of 250ml flasks that baffle plate is housed that are equipped with the 50ml LB substratum that contains 100 μ g/ml penbritins therein
6000.6, and after wherein adding 1mM IPTG, continuing cultivation 4 hours.PT7-7 (TMLA) is separated from culture, and, carry out agarose gel electrophoresis then with NdeI and SalI digestion.The result shows in Figure 12.As shown in Figure 12, confirmation and NdeI and the corresponding band of SalI fragment (swimming lane 3).Then, separate pT7-7 (TMLA), and analyze the nucleotide sequence of TMLA.As a result, the nucleotide sequence of TMLA confirms it is the sequence (SEQ ID NO:18) identical with the sequence of preserving in the Neuraspora crassa genome database of NCBI.
In addition, in the culture of the e. coli bl21 (DE3) that transforms with pT7-7 (TMLA), confirm the TMLA albumen of expression.At first, 4, centrifugal 15 minutes of 000xg carries out centrifugation to culture, and the collecting cell precipitation.The lysis buffer (140mM NaCl, 200g/l glycerine and 1mM DTT are in the buffer solution of sodium phosphate of 10mM pH 7.4) of adding 1ml and resuspended in the cell precipitation that obtains.Cell suspension is placed ice bath, and cell is used the sonicator cracking by transmitting ultrasonic wave 5 times each 10 seconds.The pair cell lysate carries out centrifugation, at 4 ℃, and 10, the centrifugal 20-30 of 000g minute, remove cell debris then, and collect supernatant liquor to obtain cell crude extract.The sample that comes from the cell crude extract of acquisition by collecting carries out 8%SDS-PAGE (referring to Figure 13, swimming lane 3).As the result who carries out SDS-PAGE, confirm band with the corresponding about 53kDa of TMLA.
(4) the multinuclear glycosides of amplification and clones coding γ-trimethylammonium amino-aldehyde desaturase (TMABADH)
The generation of acid and confirmation TMABADH
(a) the multinuclear glycosides of amplification and clones coding γ-trimethylammonium amino-aldehyde desaturase (TMABADH)
Acid
Use comprises the λ of cDNA library set of (1) as template, and uses SEQ ID NOS:7 and 8 to carry out PCR as primer.Then, carry out agarose gel electrophoresis for the PCR product that obtains.As a result, confirm the product that needs of about 1.5kb.SEQ ID NOS:7 and 8 primer comprise that coding derives from the initiator codon of TMABDH of Neuraspora crassa and the sequence of terminator codon.By from the aminoacid sequence of the whole protein of Neuraspora crassa genomic expression and derive from the people and the aminoacid sequence of the known TMABADH of rat between carry out the potential TMABADH that homology search derives from Neuraspora crassa, and from the aminoacid sequence design primer SEQ ID NOS:7 and 8 of potential TMABADH.With the PCR product with EcoRI and SalI digestion, and with pBS KS with identical enzymic digestion
+(Stratagene Inc.) connects, then with the bacillus coli DH 5 alpha pBS KS that wherein inserts the PCR product that is obtained
+(TMABADH) transform.The bacillus coli DH 5 alpha that transforms was cultivated 8 hours at 37 ℃, therefrom separated pBS KS then
+And, whether correctly insert (TMABADH), to determine the PCR product with EcoRI and SalI digestion.Then, with isolating pBS KS
+(TMABADH) with NdeI and SalI digestion, after agarose gel electrophoresis, NdeI is separated with the SalI fragment then.With described fragment with connect with the expression vector pT7-7 of identical enzymic digestion, to obtain pT7-7 (TMABADH) (referring to Figure 10).E. coli bl21 (DE3) is transformed with pT7-7 (TMABADH).
(b) confirm that TMABADH produces
The e. coli bl21 (DE3) that will transform with the pT7-7 (TMABADH) that obtains is cultured to OD in 37 ℃ of 250ml flasks that baffle plate is housed that are equipped with the 50ml LB substratum that contains penbritin therein
6000.6, and after wherein adding 1mM IPTG, continuing cultivation 4 hours.PT7-7 (TMABADH) is separated from culture, and, carry out agarose gel electrophoresis then with NdeI and SalI digestion.The result shows in Figure 12.As shown in Figure 12, confirmation and NdeI and the corresponding band of SalI fragment (swimming lane 4).Then, separate pT7-7 (TMABADH), and analyze the nucleotide sequence of TMLH.As a result, the nucleotide sequence of TMABADH confirms it is the sequence (SEQ ID NO:19) identical with the sequence of preserving in the Neuraspora crassa genome database of NCBI.
In addition, in the culture of the e. coli bl21 (DE3) that transforms with pT7-7 (TMABADH), confirm the TMABADH albumen of expression.At first, culture is carried out centrifugation, 4, centrifugal 15 minutes of 000xg, and collecting cell precipitation.The lysis buffer (140mM NaCl, 200g/l glycerine and 1mM DTT are in the buffer solution of sodium phosphate of 10mM pH 7.4) of adding 1ml and resuspended in the cell precipitation that obtains.Cell suspension is placed ice bath, and cell is used the sonicator cracking by transmitting ultrasonic wave 5 times each 10 seconds.The pair cell lysate carries out centrifugation, at 4 ℃, and 10, the centrifugal 20-30 of 000g minute, remove cell debris then, and collect supernatant liquor to obtain cell crude extract.The sample that comes from the cell crude extract of acquisition by collecting carries out 8%SDS-PAGE (referring to Figure 13).As the result who carries out SDS-PAGE, confirm band with the corresponding about 55kD of TMABADH.
(5) polynucleotide and the card of amplification and clones coding gamma-butyrobetaine hydroxylase (BBH)
The generation of real BBH
(a) polynucleotide of amplification and clones coding gamma-butyrobetaine hydroxylase (BBH)
Use comprises the λ of cDNA library set of (1) as template, and uses SEQ ID NOS:9 and 10 to carry out PCR as primer.Then, carry out agarose gel electrophoresis for the PCR product that obtains.As a result, confirm the product that needs of about 1.3kb.SEQ ID NOS:9 and 10 primer comprise that the supposition coding derives from the initiator codon of BBH of Neuraspora crassa and the sequence of terminator codon.By from the aminoacid sequence of the whole protein of Neuraspora crassa genomic expression and derive from the people and the aminoacid sequence of the known BBH of rat between carry out the potential BBH that homology search derives from Neuraspora crassa, and from the aminoacid sequence design primer SEQ IDNOS:9 and 10 of potential BBH.
The PCR product with EcoRI and SalI digestion, and is connected with pUC19 with identical enzymic digestion, then with bacillus coli DH 5 alpha with pUC19 (BBH) conversion of wherein inserting the PCR product that is obtained.The bacillus coli DH 5 alpha that transforms was cultivated 8 hours at 37 ℃ in the LB substratum that comprises 100 μ g/ml penbritins, therefrom separated pUC19 (BBH) then, and, whether correctly insert to determine the PCR product with EcoRI and SalI digestion.Then, isolating pUC 19 (BBH) with NdeI and SalI digestion, is separated NdeI after agarose gel electrophoresis then with the SalI fragment.With described fragment with connect with the expression vector pT7-7 of identical enzymic digestion, to obtain pT7-7 (BBH) (referring to Figure 11).E. coli bl21 (DE3) is transformed with pT7-7 (BBH).
The e. coli bl21 (DE3) that will transform with the pT7-7 (BBH) that obtains is cultured to OD in 37 ℃ of 250ml flasks that baffle plate is housed that are equipped with the 50ml LB substratum that contains 100 μ g/ml penbritins therein
6000.6, and after wherein adding 1mM IPTG, continuing cultivation 4 hours.PT7-7 (BBH) is separated from culture, and digest, carry out 0.8% agarose gel electrophoresis then with NdeI and SalI.The result shows in Figure 12.As shown in Figure 12, confirmation and NdeI and the corresponding band of SalI fragment (swimming lane 5).Then, separate pT7-7 (BBH), and analyze the nucleotide sequence of BBH.As a result, the nucleotide sequence of BBH confirms it is the sequence (SEQ ID NO:20) identical with the sequence of preserving in the Neuraspora crassa genome database of NCBI.
(b) confirm the proteic generation of BBH
In the culture of the e. coli bl21 (DE3) that transforms with pT7-7 (BBH), confirm the BBH albumen of expression.At first, 4, centrifugal 15 minutes of 000xg carries out centrifugation to culture, and the collecting cell precipitation.The lysis buffer (140mMNaCl, 200g/l glycerine and 1mM DTT are in the buffer solution of sodium phosphate of 10mM pH 7.4) of adding 1ml and resuspended in the cell precipitation that obtains.Cell suspension is placed ice bath, and cell is used the sonicator cracking by transmitting ultrasonic wave 5 times each 10 seconds.The pair cell lysate carries out centrifugation, at 4 ℃, and 10, the centrifugal 20-30 of 000g minute, remove cell debris then, and collect supernatant liquor to obtain cell crude extract.The sample that comes from the cell crude extract of acquisition by collecting carries out 8%SDS-PAGE (referring to Figure 13, swimming lane 5).As the result who carries out SDS-PAGE, confirm band with the corresponding about 49kDa of BBH.
Embodiment 2: produce comprise all LMT, TMLH, TMLA, TMABADH and
The host cell of BBH gene
Amplification derives from LMT, TMLH and the BBH gene in the Neuraspora crassa cDNA library that produces in embodiment 1, and produces the pT7-7 ABE with all these three kinds of genes.In addition, produce TMLA and TMABADH gene, and produce pACYC184-CarCD with all these two kinds of genes from the Neuraspora crassa cDNA library that produces among the embodiment 1.The pT7-7-CarABE and the pACYC184-CarCD that are produced are used for intestinal bacteria, have the microorganism transformed of all LMT, TMLH, TMLA, TMABADH and BBH gene with generation.Institute's microorganism transformed is called e. coli bl21 (DE3) CJ2004-2, and be deposited in Korea S microbial preservation center (Korean Culture Center ofMicroorganisms on December 13rd, 2004, KCCM), an international preservation mechanism (preserving number KCCM-10638).
(1) produces pT7-7-CarABE with all three kinds of gene LMT, TMLH and BBH
At first, use the cDNA library that derives from Neuraspora crassa, and the oligonucleotide that uses SEQID NOS:1 and 2 comprise the 1mt of terminator codon as primer from the amplification of T7 promotor as template.Then, use the cDNA library that derives from Neuraspora crassa, and the oligonucleotide that uses SEQ ID NOS:3 and 4 comprise the TMLH of terminator codon as primer from the amplification of T7 promotor as template.Then, use oligonucleotide SEQ ID NOS:9 and 10, comprise the BBH of terminator codon from the amplification of T7 promotor as primer.The amplified production of LMT, TMLH and BBH is incorporated among the pT7-7.At first, the amplified production of BBH with Restriction Enzyme such as BamHI and SalI digestion, and is therefrom obtained BamHI and SalI fragment, then, with described fragment and pT7-7 connection, with acquisition pT7-7 BBH with identical enzymic digestion.Then, the amplified production of TMLH with NdeI and EcoRI digestion, and is therefrom obtained NdeI and EcoRI fragment, then, with described fragment and pT7-7 BBH connection, with acquisition pT7-7 CarBE with identical enzymic digestion.Then, the amplified production of LMT is digested with ClaI, and therefrom obtain the ClaI fragment, then, described fragment is connected with 1mt with identical enzymic digestion, to obtain pT7-7 CarABE (referring to Figure 14).
(2) produce pACYC184 CarCD with all gene TMLA and TMABADH
At first, use the cDNA library that derives from Neuraspora crassa, and the oligonucleotide that uses SEQID NOS:5 and 6 comprise the TMLA of terminator codon as primer from the amplification of T7 promotor as template.Then, use the cDNA library that derives from Neuraspora crassa, and the oligonucleotide that uses SEQ ID NOS:7 and 8 comprise the TMABADH of terminator codon as primer from the amplification of T7 promotor as template.The amplified production of TMLA and TMABADH is incorporated among the pACYC184.At first, the amplified production of TMLA with BamHI and HindIII digestion, and is therefrom obtained BamHI and HindIII fragment, then, with described fragment and pACYC184 connection, with acquisition pACYC184 TMLA with identical enzymic digestion.Then, the amplified production of TMABADH with BamHI and SalI digestion, and is therefrom obtained BamHI and SalI fragment, then, with described fragment and pACYC184 TMLA connection, with acquisition pACYC184 CarCD with identical enzymic digestion.
Embodiment 3: use comprise coding LMT, TMLH, TMLA, TMABADH and
The microorganisms producing L-carnitine of the polynucleotide of BBH
Cultivate in the substratum that contains L-Methionin wherein being introduced among the embodiment 2 the two e. coli bl21 (DE3) of the pT7-7-CarABE that produces and pACYC184-CarCD, and measure the output of L-carnitine.PT7-7-CarABE and pACYC184-CarCD introduce e. coli bl21 (DE3) application and carry out as embodiment 1 described electroporation.
(1) by cultivation be used among the embodiment 2 pT7-7-CarABE that produces and
The e. coli bl21 of the two conversion of pACYC184-CarCD (DE3) and produce the L-carnitine
At first, will be seeded in the e. coli bl21 (DE3) of pT7-7-CarABE and the two conversion of pACYC184-CarCD in the LB solid plate substratum that contains penbritin (100 μ g/ml) and paraxin (50 μ g/ml), and cultivate.To drop at the microbial bacteria on the solid plate substratum in the flask that contains 20ml LB substratum and cultivate 12 hours to OD at 37 ℃
6001.0, add penbritin (100 μ g/ml) and paraxin (50 μ g/ml) in the wherein said LB substratum.The culture of 0.1ml institute cultured microorganism is inoculated in the 250ml flask that baffle plate is housed, wherein contains 20ml LB substratum, and then be cultured to OD at 37 ℃ with 2mM L-Methionin
6000.6.When adding IPTG, at OD
600Value reaches 0.6 back and adds 1mM IPTG, then microorganism is continued to cultivate 4 hours.To in the LB of no L-Methionin substratum, cultivate described microorganism and use same procedure mentioned above with IPTG inductive group with containing in the LB substratum of L-Methionin and cultivate described microorganism, as control group without IPTG inductive group.After cultivating termination, use the L-carnitine content of determining culture as identical method in (1).The result is displayed in Table 2.
Table 2. is produced the L-carnitine by monoculture
| Culture condition | Concentration (mg/l) |
| LB substratum (IPTG induces) | 0 |
| The LB substratum (no IPTG induces) that contains 2mM Methionin | 0.14 |
| The LB substratum (IPTG induces) that contains 2mM Methionin | 19.81 |
As shown in table 2, by in the substratum that contains L-Methionin, cultivating the microorganism of all polynucleotide that contain encode LMT, TMLH, TMLA, TMABADH and BBH, can be with high efficiency production L-carnitine.In addition, in table 2, compare mutually between the L-carnitine output of expression, and confirm that the L-carnitine has higher productive rate when cultivating in the substratum that is containing Methionin.
| Applicant or agency's file references | International application no |
Relate to the microorganism of preservation or the indication of other biomaterial
(PCT detailed rules and regulations 13bis)
PCR/RO/134 shows (in July, 1998)
Sequence table
＜110〉CJ Co., Ltd.
＜120〉carry with the enterobacteriaceae microorganism belonging to genus of L-carnitine biosynthesis related genes and use the method for this microorganisms producing L-carnitine
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＜213〉artificial sequence
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＜223〉primer
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ggaattccat atggccttcg gaaagcttta cac 33
<210>2
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
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cgggatcctt agacgttggt caacttgggg 30
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＜213〉artificial sequence
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atgaattcca tatgagaccg caagtggtag gg 32
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＜213〉artificial sequence
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＜213〉artificial sequence
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attagtcgac tcaataccct cccccaccctg 31
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<212>PRT
＜213〉Neuraspora crassa (Neurospora crassa)
<400>11
Met Ala Phe Gly Lys Leu Tyr Thr Tyr Glu Ala Asn Pro Arg Ser Thr
1 5 10 15
Ala Ile Leu Ala Val Ala Lys Ala Asn Asn Leu Asp Leu Glu Val Ile
20 25 30
Lys Val Asp Leu Glu Ala Ala Ile Glu Glu Tyr Lys Lys Val Asn Pro
35 40 45
Leu Gly Lys Val Pro Thr Phe Val Gly Ala Asp Gly Tyr Thr Leu Phe
50 55 60
Glu Cys Ile Ala Ile Ala Ile Tyr Val Ala Ser Gln Asn Glu Lys Thr
65 70 75 80
Thr Leu Leu Gly Lys Thr Lys Gln Asp Tyr Ala Ser Ile Leu Lys Trp
85 90 95
Leu Ser Phe Phe Asn Thr Glu Val Leu Pro Pro Leu Ala Gly Trp Tyr
100 105 110
Arg Pro Leu Leu Gly Lys Ala Pro Tyr Asn Lys Lys Ala Val Glu Asp
115 120 125
Ala Gln Ala Thr Ala Leu Lys Ala Ile Ser Val Ala Glu Ala His Leu
130 135 140
Lys Asn Asn Thr Phe Pro Val Gly Glu Arg Ile Thr Leu Ala Asp Leu
145 150 155 160
Phe Ala Thr Gly Ile Ile Ala Arg Gly Phe Glu Phe Phe Phe Asp Lys
165 170 175
Ala Trp Arg Glu Gln Tyr Pro Asn Val Thr Arg Trp Tyr Thr Thr Val
180 185 190
Tyr Asn Gln Pro Ile Tyr Ser Ala Val Ala Pro Pro Phe Ala Leu Leu
195 200 205
Asp Thr Pro Lys Leu Thr Asn Val
210 215
<210>12
<211>471
<212>PRT
＜213〉Neuraspora crassa
<400>12
Met Arg Pro Gln Val Val Gly Ala Ile Leu Arg Ser Arg Ala Val Val
1 5 10 15
Ser Arg Gln Pro Leu Ser Arg Thr His Ile Phe Ala Ala Val Thr Val
20 25 30
Ala Lys Ser Ser Ser Pro Ala Gln Asn Ser Arg Arg Thr Phe Ser Ser
35 40 45
Ser Phe Arg Arg Leu Tyr Glu Pro Lys Ala Glu Ile Thr Ala Glu Gly
50 55 60
Leu Glu Leu Ser Pro Pro Gln Ala Val Thr Gly Gly Lys Arg Thr Val
65 70 75 80
Leu Pro Asn Phe Trp Leu Arg Asp Asn Cys Arg Cys Thr Lys Cys Val
85 90 95
Asn Gln Asp Thr Leu Gln Arg Asn Phe Asn Thr Phe Ala Ile Pro Ser
100 105 110
Asp Ile His Pro Thr Lys Val Glu Ala Thr Lys Glu Asn Val Thr Val
115 120 125
Gln Trp Ser Asp Asn His Thr Ser Thr Tyr Pro Trp Pro Phe Leu Ser
130 135 140
Phe Tyr Leu Thr Ser Asn Ala Arg Gly His Glu Asn Asp Gln Ile Ser
145 150 155 160
Leu Trp Gly Ser Glu Ala Gly Ser Arg Pro Pro Thr Val Pro Phe Pro
165 170 175
Arg Val Met Ala Ser Asp Gln Gly Val Ala Asp Leu Thr Ala Met Ile
180 185 190
Lys Glu Phe Gly Phe Cys Phe Val Lys Asp Thr Pro His Asp Asp Pro
195 200 205
Asp Val Thr Arg Gln Leu Leu Glu Arg Ile Ala Phe Ile Arg Val Thr
210 215 220
His Tyr Gly Gly Phe Tyr Asp Phe Thr Pro Asp Leu Ala Met Ala Asp
225 230 235 240
Thr Ala Tyr Thr Asn Leu Ala Leu Pro Ala His Thr Asp Thr Thr Tyr
245 250 255
Phe Thr Asp Pro Ala Gly Leu Gln Ala Phe His Leu Leu Glu His Lys
260 265 270
Ala Ala Pro Ser Arg Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro
275 280 285
Ser Glu Glu Lys Glu Ala Ala Gly Ser Ala Ala Gly Glu Ala Ala Ala
290 295 300
Ala Ala Glu Gly Gly Lys Ser Leu Leu Val Asp Gly Phe Asn Ala Ala
305 310 315 320
Arg Ile Leu Lys Glu Glu Asp Pro Arg Ala Tyr Glu Ile Leu Ser Ser
325 330 335
Val Arg Leu Pro Trp His Ala Ser Gly Asn Glu Gly Ile Thr Ile Ala
340 345 350
Pro Asp Lys Leu Tyr Pro Val Leu Glu Leu Asn Glu Asp Thr Gly Glu
355 360 365
Leu His Arg Val Arg Trp Asn Asn Asp Asp Arg Gly Val Val Pro Phe
370 375 380
Gly Glu Lys Tyr Ser Pro Ser Glu Trp Tyr Glu Ala Ala Arg Lys Trp
385 390 395 400
Asp Gly Ile Leu Arg Arg Lys Ser Ser Glu Leu Trp Val Gln Leu Glu
405 410 415
Pro Gly Lys Pro Leu Ile Phe Asp Asn Trp Arg Val Leu His Gly Arg
420 425 430
Ser Ala Phe Ser Gly Ile Arg Arg Ile Cys Gly Gly Tyr Ile Asn Arg
435 440 445
Asp Asp Phe Ile Ser Arg Trp Arg Asn Thr Asn Tyr Pro Arg Ser Glu
450 455 460
Val Leu Pro Arg Val Thr Gly
465 470
<210>13
<211>480
<212>PRT
＜213〉Neuraspora crassa
<400>13
Met Ser Thr Tyr Ser Leu Ser Glu Thr His Lys Ala Met Leu Glu His
1 5 10 15
Ser Leu Val Glu Ser Asp Pro Gln Val Ala Glu Ile Met Lys Lys Glu
20 25 30
Val Gln Arg Gln Arg Glu Ser Ile Ile Leu Ile Ala Ser Glu Asn Val
35 40 45
Thr Ser Arg Ala Val Phe Asp Ala Leu Gly Ser Pro Met Ser Asn Lys
50 55 60
Tyr Ser Glu Gly Leu Pro Gly Ala Arg Tyr Tyr Gly Gly Asn Gln His
65 70 75 80
Ile Asp Glu Ile Glu Val Leu Cys Gln Asn Arg Ala Leu Glu Ala Phe
85 90 95
His Leu Asp Pro Lys Gln Trp Gly Val Asn Val Gln Cys Leu Ser Gly
100 105 110
Ser Pro Ala Asn Leu Gln Val Tyr Gln Ala Ile Met Pro Val His Gly
115 120 125
Arg Leu Met Gly Leu Asp Leu Pro His Gly Gly His Leu Ser His Gly
130 135 140
Tyr Gln Thr Pro Gln Arg Lys Ile Ser Ala Val Ser Thr Tyr Phe Glu
145 150 155 160
Thr Met Pro Tyr Arg Val Asn Ile Asp Thr Gly Leu Ile Asp Tyr Asp
165 170 175
Thr Leu Glu Lys Asn Ala Gln Leu Phe Arg Pro Lys Val Leu Val Ala
180 185 190
Gly Thr Ser Ala Tyr Cys Arg Leu Ile Asp Tyr Glu Arg Met Arg Lys
195 200 205
Ile Ala Asp Ser Val Gly Ala Tyr Leu Val Val Asp Met Ala His Ile
210 215 220
Ser Gly Leu Ile Ala Ser Glu Val Ile Pro Ser Pro Phe Leu Tyr Ala
225 230 235 240
Asp Val Val Thr Thr Thr Thr His Lys Ser Leu Arg Gly Pro Arg Gly
245 250 255
Ala Met Ile Phe Phe Arg Arg Gly Val Arg Ser Val Asp Ala Lys Thr
260 265 270
Gly Lys Glu Thr Leu Tyr Asp Leu Glu Asp Lys Ile Asn Phe Ser Val
275 280 285
Phe Pro Gly His Gln Gly Gly Pro His Asn His Thr Ile Thr Ala Leu
290 295 300
Ala Val Ala Leu Lys Gln Ala Ala Ser Pro Glu Phe Lys Glu Tyr Gln
305 310 315 320
Gln Lys Val Val Ala Asn Ala Lys Ala Leu Glu Lys Lys Leu Lys Glu
325 330 335
Leu Gly Tyr Lys Leu Val Ser Asp Gly Thr Asp Ser His Met Val Leu
340 345 350
Val Asp Leu Arg Pro Ile Gly Val Asp Gly Ala Arg Val Glu Phe Leu
355 360 365
Leu Glu Gln Ile Asn Ile Thr Cys Asn Lys Asn Ala Val Pro Gly Asp
370 375 380
Lys Ser Ala Leu Thr Pro Gly Gly Leu Arg Ile Gly Thr Pro Ala Met
385 390 395 400
Thr Ser Arg Gly Phe Gly Glu Ala Asp Phe Glu Lys Val Ala Val Phe
405 410 415
Val Asp Glu Ala Val Lys Leu Cys Lys Glu Ile Gln Ala Ser Leu Pro
420 425 430
Lys Glu Ala Asn Lys Gln Lys Asp Phe Lys Ala Lys Ile Ala Thr Ser
435 440 445
Asp Ile Pro Arg Ile Asn Glu Leu Lys Gln Glu Ile Ala Ala Trp Ser
450 455 460
Asn Thr Phe Pro Leu Pro Val Glu Gly Trp Arg Tyr Asp Ala Gly Leu
465 470 475 480
<210>14
<211>495
<212>PRT
＜213〉Neuraspora crassa
<400>14
Met Glu Val Glu Leu Thr Ala Pro Asn Gly Lys Lys Trp Met Gln Pro
1 5 10 15
Leu Gly Leu Phe Ile Asn Asn Glu Phe Val Lys Ser Ala Asn Glu Gln
20 25 30
Lys Leu Ile Ser Ile Asn Pro Thr Thr Glu Glu Glu Ile Cys Ser Val
35 40 45
Tyr Ala Ala Thr Ala Glu Asp Val Asp Ala Ala Val Ser Ala Ala Arg
50 55 60
Lys Ala Phe Arg His Glu Ser Trp Lys Ser Leu Ser Gly Thr Glu Arg
65 70 75 80
Gly Ala Leu Met Arg Lys Leu Ala Asp Leu Val Ala Glu Asn Ala Glu
85 90 95
Ile Leu Ala Thr Ile Glu Cys Leu Asp Asn Gly Lys Pro Tyr Gln Thr
100 105 110
Ala Leu Asn Glu Asn Val Pro Glu Val Ile Asn Val Leu Arg Tyr Tyr
115 120 125
Ala Gly Tyr Ala Asp Lys Asn Phe Gly Gln Val Ile Asp Val Gly Pro
130 135 140
Ala Lys Phe Ala Tyr Thr Val Lys Glu Pro Leu Gly Val Cys Gly Gln
145 150 155 160
Ile Ile Pro Trp Asn Tyr Pro Leu Asp Met Ala Ala Trp Lys Leu Gly
165 170 175
Pro Ala Leu Cys Cys Gly Asn Thr Val Val Leu Lys Leu Ala Glu Gln
180 185 190
Thr Pro Leu Ser Val Leu Tyr Leu Ala Lys Leu Ile Lys Glu Ala Gly
195 200 205
Phe Pro Pro Gly Val Ile Asn Ile Ile Asn Gly His Gly Arg Glu Ala
210 215 220
Gly Ala Ala Leu Val Gln His Pro Gln Val Asp Lys Ile Ala Phe Thr
225 230 235 240
Gly Ser Thr Thr Thr Gly Lys Glu Ile Met Lys Met Ala Ser Tyr Thr
245 250 255
Met Lys Asn Ile Thr Leu Glu Thr Gly Gly Lys Ser Pro Leu Ile Val
260 265 270
Phe Glu Asp Ala Asp Leu Glu Leu Ala Ala Thr Trp Ser His Ile Gly
275 280 285
Ile Met Ser Asn Gln Gly Gln Ile Cys Thr Ala Thr Ser Arg Ile Leu
290 295 300
Val His Glu Lys Ile Tyr Asp Glu Phe Val Glu Lys Phe Lys Ala Lys
305 310 315 320
Val Gln Glu Val Ser Val Leu Gly Asp Pro Phe Glu Glu Ser Thr Phe
325 330 335
His Gly Pro Gln Val Thr Lys Ala Gln Tyr Glu Arg Val Leu Gly Tyr
340 345 350
Ile Asn Val Gly Lys Glu Glu Gly Ala Thr Val Met Met Gly Gly Glu
355 360 365
Pro Ala Pro Gln Asn Gly Lys Gly Phe Phe Val Ala Pro Thr Val Phe
370 375 380
Thr Asn Val Lys Pro Thr Met Lys Ile Phe Arg Glu Glu Ile Phe Gly
385 390 395 400
Pro Cys Val Ala Ile Thr Thr Phe Lys Thr Glu Glu Glu Ala Leu Thr
405 410 415
Leu Ala Asn Asp Ser Met Tyr Gly Leu Gly Ala Ala Leu Phe Thr Lys
420 425 430
Asp Leu Thr Arg Ala His Arg Val Ala Arg Glu Ile Glu Ala Gly Met
435 440 445
Val Trp Val Asn Ser Ser Asn Asp Ser Asp Phe Arg Ile Pro Phe Gly
450 455 460
Gly Val Lys Gln Ser Gly Ile Gly Arg Glu Leu Gly Glu Ala Gly Leu
465 470 475 480
Ala Pro Tyr Cys Asn Val Lys Ser Ile His Val Asn Leu Ala Ala
485 490 495
<210>15
<211>425
<212>PRT
＜213〉Neuraspora crassa
<400>15
Met Ala Thr Ala Ala Val Gln Val Ser Val Pro Ala Pro Val Gly Gln
1 5 10 15
Pro Asp Ile Gly Tyr Ala Pro Asp His Asp Lys Tyr Leu Ala Arg Val
20 25 30
Lys Arg Arg Arg Glu Asn Glu Lys Leu Glu Ser Ser Leu Pro Pro Gly
35 40 45
Phe Pro Arg Arg Leu Asp Ser Asp Leu Val Trp Asp Gly Asn Thr Leu
50 55 60
Ala Glu Thr Tyr Asp Trp Thr Tyr Arg Leu Thr Glu Glu Ala Ile Asp
65 70 75 80
Glu Ile Glu Ala Ala Leu Arg His Phe Lys Ser Leu Asn Lys Pro Leu
85 90 95
Gly Tyr Ile Asn Gln Glu Thr Phe Pro Leu Pro Arg Leu His His Thr
100 105 110
Leu Arg Ser Leu Ser His Glu Leu His His Gly His Gly Phe Lys Val
115 120 125
Leu Arg Gly Leu Pro Val Thr Ser His Thr Arg Glu Glu Asn Ile Ile
130 135 140
Ile Tyr Ala Gly Val Ser Ser His Val Ala Pro Ile Arg Gly Arg Gln
145 150 155 160
Asp Asn Gln His Asn Gly His Pro Ala Asp Val Val Leu Ala His Ile
165 170 175
Lys Asp Leu Ser Thr Thr Val Ser Asp Val Ser Lys Ile Gly Ala Pro
180 185 190
Ala Tyr Thr Thr Glu Lys Gln Val Phe His Thr Asp Ala Gly Asp Ile
195 200 205
Val Ala Leu Phe Cys Leu Gly Glu Ala Ala Glu Gly Gly Gln Ser Tyr
210 215 220
Leu Ser Ser Ser Trp Lys Val Tyr Asn Glu Leu Ala Ala Thr Arg Pro
225 230 235 240
Asp Leu Val Arg Thr Leu Ala Glu Pro Trp Val Ala Asp Glu Phe Gly
245 250 255
Lys Glu Gly Arg Lys Phe Ser Val Arg Pro Leu Leu His Phe Gln Ser
260 265 270
Thr Ala Ala Ala Ala Ser Arg Glu Ala Lys Pro Glu Ser Glu Arg Leu
275 280 285
Ile Ile Gln Tyr Ala Arg Arg Thr Phe Thr Gly Tyr Trp Gly Leu Pro
290 295 300
Arg Ser Ala Asp Ile Pro Pro Ile Thr Glu Ala Gln Ala Glu Ala Leu
305 310 315 320
Asp Ala Leu His Phe Thr Ala Glu Lys Tyr Ala Val Ala Leu Asp Phe
325 330 335
Arg Gln Gly Asp Val Gln Phe Val Asn Asn Leu Ser Val Phe His Ser
340 345 350
Arg Ala Gly Phe Arg Asp Glu Gly Glu Lys Gln Arg His Leu Val Arg
355 360 365
Leu Trp Leu Arg Asp Pro Glu Asn Ala Trp Glu Thr Pro Glu Ala Leu
370 375 380
Lys Glu Arg Trp Glu Arg Val Tyr Gly Gly Val Ser Pro Glu Arg Glu
385 390 395 400
Val Phe Pro Leu Glu Pro Gln Ile Arg Ser Ala Ser Lys Gly Glu Ser
405 410 415
Val Gly Thr Gln Gly Gly Gly Gly Tyr
420 425
<210>16
<211>651
<212>DNA
＜213〉Neuraspora crassa
<400>16
atggccttcg gaaagcttta cacctacgag gcgaaccccc gctccacggc catcttggct 60
gtcgcgaagg ccaacaacct cgacctcgag gttatcaagg tcgaccttga ggctgccatc 120
gaggagtaca agaaggtcaa ccctctcggc aaggtcccca ccttcgttgg tgccgacggc 180
tacactctct tcgagtgcat cgccatcgcc atctatgtcg cttcccagaa cgagaagacc 240
actctcctcg gcaagaccaa gcaggactat gcctccatcc tgaagtggct ctctttcttc 300
aacaccgagg tccttccccc tcttgctggc tggtaccgcc ctctccttgg caaggctccc 360
tacaacaaga aggctgttga ggacgctcag gctactgccc tcaaggccat ctctgtcgcc 420
gaggcccacc tcaagaacaa caccttcccc gttggcgagc gcatcaccct tgccgatctc 480
ttcgccactg gcatcattgc ccgcggcttc gagttcttct tcgacaaggc ctggcgcgag 540
cagtacccca acgtcacccg ttggtacacc actgtctaca accagcccat ctactcggcc 600
gttgctcctc ccttcgctct ccttgatacc cccaagttga ccaacgtcta a 651
<210>17
<211>1407
<212>DNA
＜213〉Neuraspora crassa
<400>17
atgagaccgc aagtggtagg ggcaatcctc cgctctagag ctgttgtcag cagacaacct 60
ctttcgagga cccatatctt tgctgccgtc actgttgcaa agtcctcatc acctgcccag 120
aactcgagaa gaaccttttc atcctctttc cgacggttgt atgagccaaa ggcggagata 180
acagctgagg gacttgagtt gagccctcca caggctgtta cgggtggaaa gcggactgtt 240
ttacccaact tctggctacg tgacaactgc cggtgtacga aatgcgtgaa ccaagatact 300
ctccagagaa acttcaacac ttttgccatc ccctccgaca tccacccaac aaaggttgaa 360
gccaccaagg agaacgtcac cgtccaatgg tccgacaacc acacatccac ctacccctgg 420
cccttcctct ctttctacct cacctccaac gcgcgcgggc acgaaaacga ccagatctcc 480
ctctggggct ccgaagccgg ctcccgcccg ccaaccgtct ccttccctcg cgtgatggca 540
tcagaccagg gcgtcgccga cctaaccgcc atgatcaaag agttcggctt ctgtttcgtc 600
aaagacacac cccatgacga cccggacgtg acccgccagc ttctggagag aatcgccttt 660
atccgagtga cccattacgg cggcttttac gatttcacgc ccgacctcgc gatggccgac 720
acggcgtaca cgaacctggc gctgccggcg catacggata cgacgtactt cacggacccg 780
gcggggttgc aggcttttca cttgttggag cataaggccg ctccttctcg tcctcctcct 840
cctcctcctc ctcctcctcc tccttctgaa gaaaaagaag ctgcaggctc agcagcaggg 900
gaggcggcgg cggcagcaga agggggaaag tcgttgttgg tcgatgggtt caacgccgcg 960
aggattctga aggaggagga tccccgggct tatgagatct tgagcagcgt gagactgccg 1020
tggcatgcga gtggaaacga agggatcacg attgcgcccg ataagcttta tccggtgctg 1080
gaactgaatg aggataccgg ggaactgcat agggttaggt ggaataatga tgataggggt 1140
gtggtgccgt ttggggagaa gtacagcccg tcagagtggt atgaggcggc gaggaagtgg 1200
gatgggattt tgaggaggaa gagcagcgag ttgtgggtgc agttggagcc ggggaagccg 1260
ttgaggttct tcatggacgg agcgcgttct cgggtattag gaggatttgt ggagggtata 1320
tcaaccgcga tgacttcatc tctcggtgga ggaacacgaa ttacccaagg agcgaggttc 1380
ttccgagggt tactggttaa ggactga 1407
<210>18
<211>1443
<212>DNA
＜213〉Neuraspora crassa
<400>18
atgtctacct actccctctc cgagactcac aaggccatgc tcgagcatag cttggtcgag 60
tccgaccccc aggtcgccga gatcatgaag aaggaggttc agcgccagcg cgagtccatc 120
atcctcatcg cctccgagaa cgtcacctcg cgtgccgtct tcgatgccct cggctccccc 180
atgtccaaca agtactcgga gggtcttccc ggcgcccgct actatggtgg caaccagcac 240
atcgacgaga tcgaggttct ctgccagaac cgtgcccttg aggccttcca cctcgacccc 300
aagcagtggg gtgtcaatgt tcagtgcttg tccggcagcc ctgccaacct ccaggtctac 360
caggccatca tgcccgtcca cggcagactc atgggtcttg acctccccca cggtggccat 420
ctttcccacg gttaccagac cccccagcgc aagatctctg ctgtctctac ctacttcgag 480
accatgccct accgcgtcaa cattgacact ggtctcatcg actacgatac cctcgagaag 540
aacgcccagc tcttccgccc caaggtcctc gtcgccggta cctctgccta ctgccgtctg 600
attgactacg agcgcatgcg caagattgcc gactccgttg gcgcttacct tgtcgtcgat 660
atggctcaca tttccggcct cattgcctcc gaggttatcc cctcgccctt cctctacgcc 720
gatgtcgtca ccaccaccac tcacaagtct ctccgtggcc ctcgtggcgc catgatcttc 780
ttccgccgcg gtgtccgctc cgttgacgcc aagaccggca aggagaccct ctacgacctt 840
gaggacaaga tcaacttctc cgtcttccct ggtcaccagg gtggccccca caaccacacc 900
atcaccgccc ttgccgttgc cctcaagcag gctgcctccc ccgagttcaa ggagtaccag 960
cagaaggtcg ttgccaacgc caaggctctc gagaagaagc tcaaggagct cggctacaag 1020
ctcgtctctg acggcactga ctctcacatg gtcctcgttg accttcgccc catcggcgtc 1080
gatggtgccc gtgttgagtt cctccttgag cagatcaaca ttacctgcaa caagaacgcc 1140
gttcccggcg acaagagcgc cctcaccccc ggcggtctcc gtattggtac ccccgctatg 1200
acctcccgtg gcttcggcga ggccgacttc gagaaggtcg ccgtcttcgt cgatgaggct 1260
gtcaagctct gcaaggagat ccaggcttcc ctccccaagg aggctaacaa gcagaaggac 1320
ttcaaggcca agatcgccac cagcgatatt ccccgcatca acgagctcaa gcaggagatt 1380
gccgcctgga gcaacacctt ccccctcccc gttgagggct ggagatacga tgccggtctc 1440
taa 1443
<210>19
<211>1488
<212>DNA
＜213〉Neuraspora crassa
<400>19
atggaagtcg agcttacggc ccccaacggc aagaagtgga tgcagccact gggcttgttc 60
attaataacg agtttgtcaa aagtgccaat gagcagaagt tgatttccat caacccaact 120
accgaagagg agatctgctc ggtatacgcc gcaaccgccg aggatgttga cgccgcagta 180
tcagcagccc gcaaggcctt taggcacgaa tcatggaagt cgctatccgg cactgagcgc 240
ggcgccctga tgcgcaagct ggccgaccta gtggccgaga atgccgaaat cctagccacc 300
atcgagtgcc tggacaacgg caagccgtat cagacagccc ttaacgagaa cgtgcccgaa 360
gtgatcaacg tcctcaggta ctatgccggc tatgcggaca agaactttgg ccaagtgatt 420
gacgttggcc ccgccaagtt tgcctacacg gtcaaggagc ctctcggcgt atgtggccag 480
atcatcccct ggaactaccc gctagatatg gccgcctgga agctggggcc agctctctgc 540
tgcggcaaca ccgtggtcct caagctggcc gagcagactc ccctgtccgt gttgtacttg 600
gctaagctca ttaaggaggc cggcttccct cccggtgtga tcaatatcat caacggacac 660
ggcagggaag cgggtgccgc acttgtgcaa catcctcagg tggacaagat tgcctttacc 720
ggcagcacca ctacgggcaa ggagatcatg aagatggctt cctataccat gaagaacatc 780
accctggaga ctggcggcaa gtcaccgttg atcgtgtttg aggatgccga ccttgagctg 840
gcggcgacat ggtcacacat cggcatcatg agcaaccagg gccaaatctg cacagccact 900
tcacgcattc tcgtgcacga gaagatctac gacgagtttg tcgaaaaatt caaggccaaa 960
gtccaggagg tttcggtact cggcgacccc ttcgaggaga gcacgttcca cggacctcag 1020
gtcaccaaag cgcagtatga gcgtgttctg ggctatatca atgtcggaaa ggaagagggt 1080
gccacggtga tgatgggtgg tgagccggct ccgcagaacg gtaaaggttt ctttgtggcc 1140
ccgactgtct tcacgaacgt caagccgacc atgaagatct tcagggagga gatctttggg 1200
ccctgcgtgg ccattaccac gttcaaaacg gaggaggagg cgttgacgct ggccaacgac 1260
agcatgtatg gcctgggagc ggctctgttc accaaggacc taaccagggc acacagagtg 1320
gcgcgggaga tcgaggccgg catggtctgg gtcaacagca gcaacgattc agactttagg 1380
attccatttg gaggcgtgaa gcagtctggt attgggaggg agttgggaga ggcaggtctg 1440
gcaccttatt gcaacgtcaa gagtatccat gtaaacctgg cggcatga 1488
<210>20
<211>1278
<212>DNA
＜213〉Neuraspora crassa
<400>20
atggccacgg cagcggttca ggtttcagtc ccagctccgg ttggacaacc agatatcggg 60
tacgctcctg accacgacaa gtacctcgca agagtcaaaa gacgacgaga aaacgagaag 120
ctggagtcgt ctcttccgcc aggtttccct cgaagactag actcggacct tgtgtgggac 180
ggcaacaccc tcgccgagac gtacgactgg acctacagac tgacagaaga ggccattgat 240
gaaatcgagg ccgcgcttcg tcattttaag agcctcaaca agcccctagg ctacatcaac 300
caagaaacct tcccccttcc ccgcctacac cacactctcc gctccctctc ccacgagctc 360
caccacggcc acggcttcaa agtcctccgc gggctccccg tcacctccca tacacgcgag 420
gaaaacatca tcatctacgc cggcgtctcc tcgcatgtcg ctcctatccg cggccgccag 480
gacaaccagc acaacggcca cccagccgac gtagtcctag cacacatcaa agacctgtcc 540
acgactgttt ctgacgtgag caaaatcggt gcacccgcct acaccaccga gaaacaagtc 600
ttccacaccg acgcaggcga catcgtcgcc ctcttttgct tgggagaggc cgccgagggc 660
ggacagagtt acctgtccag cagctggaag gtgtacaacg agctggcagc cactcggccc 720
gatctggttc gcacgctggc ggagccgtgg gtggcggacg agtttggcaa ggaagggagg 780
aagttttctg tgcgaccgct tttgcatttt cagtctactg ctgctgctgc ttctagggaa 840
gcaaagcccg agtctgaacg gctcatcatc cagtacgccc gccgcacgtt tacggggtat 900
tggggattac cgaggtcggc ggatatcccg cccattacgg aggcgcaggc ggaggcgttg 960
gatgcgctgc actttacggc ggagaagtac gcggtggcgc tggatttcag gcagggggat 1020
gtccagtttg tgaataactt gagtgtgttc cattcgaggg cggggtttag agatgagggg 1080
gagaagcaga ggcatttggt taggttgtgg ttgagagatc cggagaatgc gtgggagacg 1140
cccgaggcgt tgaaggaacg gtgggaacgc gtgtatggcg gggtgagtcc ggagagggag 1200
gtgtttccgc ttgagccgca gattaggagc gcgagtaagg gggagagcgt ggggacgcag 1260
ggtgggggag ggtattga 1278
<210>21
<211>5
<212>PRT
＜213〉Neuraspora crassa
<400>21
Ala Phe Gly Lys Leu
1 5
Claims (10)
1. one kind belongs to the microorganism that enterobacteriaceae (Enterobacteriacae) belongs to, and described microorganism comprises:
Coding derives from the polynucleotide of S-adenosylmethionine-6-N-Methionin-methyl transferase activity of Neuraspora crassa (Neurospora crassa);
The polynucleotide of coding 6-N-trimethyl lysine hydroxylase activity;
The polynucleotide of coding 3-hydroxyl-6-N-trimethyl lysine aldolase activity; With
The coding γ-trimethylammonium amino-aldehyde dehydrogenase activity polynucleotide with
The active polynucleotide of coding gamma-butyrobetaine hydroxylase.
2. the microorganism of claim 1, wherein said microorganism is intestinal bacteria (Escherichiacoli).
3. the microorganism of claim 1, wherein said microorganism is an intestinal bacteria (preserving number: KCCM-10638).
4. the microorganism of claim 1, wherein said coding S-adenosylmethionine-active polynucleotide of 6-N-Methionin-methyltransgerase (LMT) are polynucleotide of the aminoacid sequence of coding SEQ ID NO:11.
5. the microorganism of claim 1, the active polynucleotide of wherein said coding 6-N-trimethyl lysine hydroxylase (TMLH) are polynucleotide of the aminoacid sequence of coding SEQ ID NO:12.
6. the microorganism of claim 1, wherein said coding 3-hydroxyl-active polynucleotide of 6-N-trimethyl lysine zymohexase (TMLA) are polynucleotide of the aminoacid sequence of coding SEQ ID NO:13.
7. the microorganism of claim 1, wherein said coding γ-active polynucleotide of trimethylammonium amino-aldehyde desaturase (TMABADH) are polynucleotide of the aminoacid sequence of coding SEQ ID NO:14.
8. the microorganism of claim 1, the active polynucleotide of described coding gamma-butyrobetaine hydroxylase (BBH) are polynucleotide of the aminoacid sequence of coding SEQ ID NO:15.
9. method of producing the L-carnitine, described method comprises:
In the presence of substrate, cultivate claim 1 to the claim 8 any one microorganism in described culture, to produce the L-carnitine, described substrate is selected from the group of being made up of following: L-Methionin, the N-trimethyl lysine, beta-hydroxy-N-trimethyl lysine, γ-N-trimethylammonium amino butyraldehyde, gamma-butyrobetaine, and composition thereof.
10. the method for claim 9, the concentration of wherein said substrate is 0.1 to 10 weight % based on substratum weight, described substrate is selected from the group of being made up of following: L-Methionin, the N-trimethyl lysine, beta-hydroxy-N-trimethyl lysine, γ-N-trimethylammonium amino butyraldehyde, gamma-butyrobetaine, and composition thereof.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020050061142 | 2005-07-07 | ||
| KR10-2005-0061142 | 2005-07-07 | ||
| KR1020050061142A KR100713103B1 (en) | 2005-07-07 | 2005-07-07 | - - A microorganism of Enterobacteriacae genus haboring genes associated with L-carintine biosynthesis and method of producing L-carnitine using the microorganism |
| PCT/KR2006/002661 WO2007007987A1 (en) | 2005-07-07 | 2006-07-07 | A microorganism of enterobacteriacae genus haboring genes associated with l-carintine biosynthesis and method of producing l-carnitine using the microorganism |
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| Publication Number | Publication Date |
|---|---|
| CN101213293A true CN101213293A (en) | 2008-07-02 |
| CN101213293B CN101213293B (en) | 2012-02-15 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2006800240009A Expired - Fee Related CN101213293B (en) | 2005-07-07 | 2006-07-07 | Microorganism of enterobacteriacae genus haboring genes associated with L-carnitine biosynthesis and method of producing L-carnitine using the microorganism |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US7718414B2 (en) |
| EP (1) | EP1904620B1 (en) |
| JP (1) | JP4782831B2 (en) |
| KR (1) | KR100713103B1 (en) |
| CN (1) | CN101213293B (en) |
| WO (1) | WO2007007987A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101068925B (en) * | 2005-07-19 | 2010-05-26 | Cj第一制糖株式会社 | A microorganism of enterobacteriacae genus haboring genes associated with L-carintine biosynthesis and method of producing L-carnitine using the micro organism |
| CN104928225A (en) * | 2015-05-27 | 2015-09-23 | 武汉中科光谷绿色生物技术有限公司 | Escherichia-coli gene engineering bacterium generating L-carnitine and construction method and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111100831B (en) * | 2018-10-25 | 2022-04-05 | 中国科学院微生物研究所 | Recombinant bacterium for producing L-carnitine and construction method and application thereof |
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| FR2398046A1 (en) | 1977-07-18 | 1979-02-16 | Inst Francais Du Petrole | L-CARNITINE ENZYMATIC SYNTHESIS |
| IT1142201B (en) | 1980-06-24 | 1986-10-08 | Sigma Tau Ind Farmaceuti | PROCEDURE FOR THE ENZYMATIC PRODUCTION OF L-CARNITINA |
| JPS59192095A (en) * | 1983-04-13 | 1984-10-31 | Ajinomoto Co Inc | Preparation of l-carnitine |
| DD221905B1 (en) * | 1983-11-03 | 1987-03-18 | Univ Leipzig | PROCESS FOR THE PREPARATION OF L (-) - CARNITINE AND ITS DERIVATIVES |
| CH664374A5 (en) * | 1985-02-27 | 1988-02-29 | Lonza Ag | METHOD FOR PRODUCING L-CARNITIN BY A MICROBIOLOGICAL WAY. |
| JPS62118899A (en) * | 1985-11-19 | 1987-05-30 | Seitetsu Kagaku Co Ltd | Production of l-carnitine |
| CA2021869C (en) * | 1989-07-28 | 2000-09-05 | Frans Hoeks | Process for microbiological batch production of l-carnitine |
| KR100713104B1 (en) * | 2005-07-07 | 2007-05-02 | 씨제이 주식회사 | --6--- S-adonosylmethionine-6-N-lysine-methyltransferase from Neurospora crassa a gene encoding the same a vector and host cell containing the same and method for producing trimethyllysine using the host cell |
| ATE472600T1 (en) * | 2005-07-19 | 2010-07-15 | Cj Cheiljedang Corp | MICROORGANISM OF THE GENUS ENTEROBACTERIACAE WITH GENES RELATED TO THE BIOSYNTHESIS OF L-CARNITINE AND METHOD FOR THE PRODUCTION OF L-CARNITINE USING THE MICROORGANISM |
-
2005
- 2005-07-07 KR KR1020050061142A patent/KR100713103B1/en active IP Right Grant
-
2006
- 2006-07-07 CN CN2006800240009A patent/CN101213293B/en not_active Expired - Fee Related
- 2006-07-07 US US11/994,969 patent/US7718414B2/en not_active Expired - Fee Related
- 2006-07-07 JP JP2008519191A patent/JP4782831B2/en not_active Expired - Fee Related
- 2006-07-07 WO PCT/KR2006/002661 patent/WO2007007987A1/en active Application Filing
- 2006-07-07 EP EP06769203.8A patent/EP1904620B1/en not_active Not-in-force
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101068925B (en) * | 2005-07-19 | 2010-05-26 | Cj第一制糖株式会社 | A microorganism of enterobacteriacae genus haboring genes associated with L-carintine biosynthesis and method of producing L-carnitine using the micro organism |
| CN104928225A (en) * | 2015-05-27 | 2015-09-23 | 武汉中科光谷绿色生物技术有限公司 | Escherichia-coli gene engineering bacterium generating L-carnitine and construction method and application thereof |
| CN104928225B (en) * | 2015-05-27 | 2018-06-12 | 武汉中科光谷绿色生物技术有限公司 | A kind of Recombinant organism for producing l-carnitine and construction method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20070006965A (en) | 2007-01-12 |
| EP1904620A1 (en) | 2008-04-02 |
| CN101213293B (en) | 2012-02-15 |
| US20080171368A1 (en) | 2008-07-17 |
| EP1904620B1 (en) | 2013-06-12 |
| KR100713103B1 (en) | 2007-05-02 |
| JP4782831B2 (en) | 2011-09-28 |
| WO2007007987A1 (en) | 2007-01-18 |
| US7718414B2 (en) | 2010-05-18 |
| EP1904620A4 (en) | 2009-11-11 |
| JP2009500014A (en) | 2009-01-08 |
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